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Application of matrix-assisted laser desorption/ionization mass spectrometry to identify species of Neotropical Anopheles vectors of malaria

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Application of matrix-assisted laser desorption/ionization mass spectrometry to identify species of Neotropical Anopheles vectors of malaria

Abstract and Figures

Background Malaria control in Panama is problematic due to the high diversity of morphologically similar Anopheles mosquito species, which makes identification of vectors of human Plasmodium challenging. Strategies by Panamanian health authorities to bring malaria under control targeting Anopheles vectors could be ineffective if they tackle a misidentified species. Methods A rapid mass spectrometry identification procedure was developed to accurately and timely sort out field-collected Neotropical Anopheles mosquitoes into vector and non-vector species. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of highly-abundant proteins were generated from laboratory-reared mosquitoes using different extraction protocols, body parts, and sexes to minimize the amount of material from specimen vouchers needed and optimize the protocol for taxonomic identification. Subsequently, the mass spectra of field-collected Neotropical Anopheles mosquito species were classified using a combination of custom-made unsupervised (i.e., Principal component analysis—PCA) and supervised (i.e., Linear discriminant analysis—LDA) classification algorithms. Results Regardless of the protocol used or the mosquito species and sex, the legs contained the least intra-specific variability with enough well-preserved proteins to differentiate among distinct biological species, consistent with previous literature. After minimizing the amount of material needed from the voucher, one leg was enough to produce reliable spectra between specimens. Further, both PCA and LDA were able to classify up to 12 mosquito species, from different subgenera and seven geographically spread localities across Panama using mass spectra from one leg pair. LDA demonstrated high discriminatory power and consistency, with validation and cross-validation positive identification rates above 93% at the species level. Conclusion The selected sample processing procedure can be used to identify field-collected Anopheles species, including vectors of Plasmodium, in a short period of time, with a minimal amount of tissue and without the need of an expert mosquito taxonomist. This strategy to analyse protein spectra overcomes the drawbacks of working without a reference library to classify unknown samples. Finally, this MALDI approach can aid ongoing malaria eradication efforts in Panama and other countries with large number of mosquito’s species by improving vector surveillance in epidemic-prone sites such as indigenous Comarcas. Electronic supplementary material The online version of this article (10.1186/s12936-019-2723-0) contains supplementary material, which is available to authorized users.
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Loaizaetal. Malar J (2019) 18:95
https://doi.org/10.1186/s12936-019-2723-0
RESEARCH
Application ofmatrix-assisted laser
desorption/ionization mass spectrometry
toidentify species ofNeotropical Anopheles
vectors ofmalaria
Jose R. Loaiza1,2,3 , Alejandro Almanza1, Juan C. Rojas1, Luis Mejía1,2, Norma D. Cervantes4,
Javier E. Sanchez‑Galan1,5, Fernando Merchán6, Arnaud Grillet6,7, Matthew J. Miller8, Luis F. De León1,9
and Rolando A. Gittens1,10*
Abstract
Background: Malaria control in Panama is problematic due to the high diversity of morphologically similar Anopheles
mosquito species, which makes identification of vectors of human Plasmodium challenging. Strategies by Panama‑
nian health authorities to bring malaria under control targeting Anopheles vectors could be ineffective if they tackle a
misidentified species.
Methods: A rapid mass spectrometry identification procedure was developed to accurately and timely sort out
field‑collected Neotropical Anopheles mosquitoes into vector and non‑vector species. Matrix‑assisted laser desorp‑
tion/ionization (MALDI) mass spectra of highly‑abundant proteins were generated from laboratory‑reared mosquitoes
using different extraction protocols, body parts, and sexes to minimize the amount of material from specimen vouch‑
ers needed and optimize the protocol for taxonomic identification. Subsequently, the mass spectra of field‑collected
Neotropical Anopheles mosquito species were classified using a combination of custom‑made unsupervised (i.e.,
Principal component analysis—PCA) and supervised (i.e., Linear discriminant analysis—LDA) classification algorithms.
Results: Regardless of the protocol used or the mosquito species and sex, the legs contained the least intra‑specific
variability with enough well‑preserved proteins to differentiate among distinct biological species, consistent with pre‑
vious literature. After minimizing the amount of material needed from the voucher, one leg was enough to produce
reliable spectra between specimens. Further, both PCA and LDA were able to classify up to 12 mosquito species, from
different subgenera and seven geographically spread localities across Panama using mass spectra from one leg pair.
LDA demonstrated high discriminatory power and consistency, with validation and cross‑validation positive identifi‑
cation rates above 93% at the species level.
Conclusion: The selected sample processing procedure can be used to identify field‑collected Anopheles species,
including vectors of Plasmodium, in a short period of time, with a minimal amount of tissue and without the need of
an expert mosquito taxonomist. This strategy to analyse protein spectra overcomes the drawbacks of working with‑
out a reference library to classify unknown samples. Finally, this MALDI approach can aid ongoing malaria eradication
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/
publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Open Access
Malaria Journal
*Correspondence: rgittens@indicasat.org.pa
1 Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de
Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP),
City of Knowledge, Panama 0843‑01103, Republic of Panama
Full list of author information is available at the end of the article
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Page 2 of 12
Loaizaetal. Malar J (2019) 18:95
Background
Despite historical and ongoing eradication efforts, human
malaria transmitted by Anopheles mosquitoes continues
to be a major public health concern around the world [1].
Malaria was one of the leading causes of death during
the construction of the Panamanian Interoceanic Canal
in the early 1900s. In Panama, malaria prevalence oscil-
lated dramatically during the last 50years, with sporadic
and/or cyclical epidemics every five to 10 years [24].
Recently however, from 2001 to 2005, a malaria outbreak
was documented in indigenous territories known as
Comarcas” where a sixfold increase in the number of
cases was observed [5, 6]. is epidemic was controlled
during subsequent years, and the number of sympto-
matic cases in the country has dropped considerably
since this event. Nonetheless, malaria is still endemic in
Panama, and there is potential for future outbreaks par-
ticularly in indigenous Comarcas with health, social and
demographic disparities [3, 6].
Malaria control in Panama is done mainly through
eradication of mosquito vectors using toxic insecti-
cides. is strategy requires that the Anopheles species
responsible for transmission be promptly and accurately
identified. Nonetheless, identification in Panama is prob-
lematic due to a high number of morphologically similar
Anopheles species [7]. Control strategies to bring malaria
down targeting Anopheles vectors could be ineffective
if they tackle a misidentified non-vector species. is
is likely the case in Panamanian indigenous Comarcas
where as many as 10 Anopheles species occur in a single
locality, and where 40% of these are expected to transmit
both Plasmodium vivax and P. falciparum to humans [5,
8]. e identification of Anopheles mosquitoes in Panama
is done using traditional morphological approaches (e.g.,
dichotomic keys), but this approach requires meticu-
lous taxonomic training and a great deal of entomo-
logical expertise [9, 10]. Also, it is time consuming and
could be impractical when inspecting numerous samples
[11]. Hence, Panamanian health authorities require new
approaches to accurately and timely sort out vector from
non-vector Anopheles species.
DNA barcoding is a valid alternative to identify arthro-
pod species because it has better taxonomic resolution
than morphological approaches, even if the promise of
being less expensive has not yet materialized [12]. DNA
barcodes work well with a small amount of tissue, and do
not require prior knowledge of insect morphology [13].
However, generating DNA barcodes requires advanced
sample preparation and proper laboratory facilities to
extract, amplify and sequence nucleic acids, most of
which are rarely found in developing countries where
arthropod-borne infections like malaria prevail [14].
In recent years, matrix–assisted laser desorption/ioni-
zation (MALDI) mass spectrometry has become an alter-
native for arthropod taxonomic identification [10, 15, 16].
is method has been used effectively to study several
aspects of vector biology, including taxonomic status (i.e.,
species boundaries), pathogen infection rates and food
source identity [1721]. MALDI mass spectrometry uses
a profile of the most abundant proteins to “fingerprint”
biological samples, and thus, is conceptually similar to
DNA barcoding, but possibly cheaper on a per sample
basis. Furthermore, MALDI can generate accurate iden-
tifications in just a few hours, rather than 5 to 10days as
in the case of DNA barcoding even in “rush” cases [22].
Previous efforts with MALDI to taxonomically classify
members of family Culicidae were successful using both
laboratory-reared and field-collected specimens, and
specific body parts (e.g., thorax, cephalothorax and/or
legs) plus samples from different regions of the world [9,
10, 2325]. Yssouf etal. [9, 10] used MALDI with all six
legs to classify mosquito species from Africa, Europe and
the US while Mewara etal. [24], using the same approach,
accurately identified specimens of four different mos-
quito genera in Northern India. More recently in France,
Vega-Rúa etal. [25] designed a double entry query proto-
col with MALDI protein spectra obtained from thoraxes
and legs to improve the identification of morphologically
compromised specimens. Hence, MALDI’s accurate and
rapid identification capabilities might prove ideal to solve
the shortcomings of taxonomically classifying Anopheles
mosquitoes in Panama, thus assisting ongoing malaria
eradication efforts by improving the vector surveillance
system in indigenous Comarcas.
Here, a methodology based on previously published
extraction protocols was adjusted and assessed the accu-
racy of MALDI identification with a small portion of
tissue from the mosquito body to otherwise preserve a
specimen voucher. Different statistical procedures were
also explored to analyse and classify protein spectra from
field-collected mosquitoes, which are difficult to evalu-
ate with currently available strategies from commercial
vendors, including working without a reference library of
well curated protein spectra. Specifically, the authors ask
efforts in Panama and other countries with large number of mosquito’s species by improving vector surveillance in
epidemic‑prone sites such as indigenous Comarcas.
Keywords: Anopheles mosquito, Taxonomic identification, MALDI, Mass spectrometry, Malaria vector, Panama
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Loaizaetal. Malar J (2019) 18:95
if MALDI mass spectrometry can discriminate among
field-collected individuals of 11 known mosquito species
in the genus Anopheles, including taxa that are vectors of
human Plasmodium in Panama, plus Chagasia bathana,
a closely phylogenetically related and ancestral species to
Anopheles.
Methods
Sample preparation andoptimization
Initial experiments were conducted with laboratory-
reared mosquitoes from three discrete biological species:
Anopheles albimanus (vector of malaria); Aedes aegypti
(vector of Zika and dengue); and Aedes albopictus (vec-
tor of Chikungunya) (Additional file1). ree different
sample preparation protocols (i.e., protein extraction
methods), adapted with minor modifications from previ-
ous studies [10, 20], were compared and the one with the
most suitable results got selected for further experimen-
tation with field-collected specimens (see the full descrip-
tion of these protocols in Table1). To test for differences
in the mass spectra produced with the three extraction
protocols, whole insect-bodies of freshly emerged and
starved female mosquitoes were used to avoid noise in
the acquired protein signal. Two hundred and twenty-
five female mosquitoes were used in total at this point,
25 individuals per species for each protocol. Further, dif-
ferent parts of the body of female mosquitoes (e.g., head,
thorax, abdomen, wings and one of the anterior, middle
and posterior legs) were assessed to confirm if they con-
tained different protein spectra, and if these spectra were
consistent across specimens of the same taxon as it has
been shown previously [9, 10, 2325]. Body parts were
dissected using a micro-dissecting kit, placed in sepa-
rate micro-centrifuge tubes and labeled accordingly. For
this evaluation, another 25 lab-reared female individu-
als of A. albimanus, A. aegypti and A. albopictus (Addi-
tional files 2 and 3) were used. Finally, the section of the
body with the highest and most consistent protein signal
was selected and proceeded to compare whether or not
females and males of a given species display differences
in their protein spectra as shown by previous studies
using whole insect bodies [20]. Once more, differences
between females and males were evaluated using 25 labo-
ratory-reared specimens of A. albimanus, A. aegypti and
A. albopictus, respectively (Additional file4).
Field‑collected Neotropical Anopheles species
For the second part of the study, fresh Anopheles mos-
quitoes from four subgenera and seven geographi-
cally spread localities in indigenous Comarcas across
Panama (Table 2) were collected. Mosquitoes were
collected at night during seven consecutive days per
location, using different types of traps (e.g., Human
Landing Catch, Intersection, Shannon and Center for
Disease Control—CDC—miniature light trap) (Addi-
tional file 5). Samples were stored at room tempera-
ture in individual, dry microtubes along with silica gel,
and transported back to the laboratory in plastic bags.
Once in the laboratory, mosquitoes were maintained
at 20°C to preserve the integrity of their proteins.
Initially, all field-collected specimens were sorted and
identified to species level using a taxonomic key based
on morphological characters of the female [26]. en,
between ten and 66 individuals per species were pro-
cessed and analysed using mass spectrometry, for a
total of 12 species and 299 specimens (Table 2). For
this section of the study, and upon analysing the out-
comes of experiments performed during the first part
Table 1 Description ofthree dierent MALDI mass spectrometry protein-extraction protocols used inthepresent study
Protocol #1: Formic acid/ethanol extraction protocol recommended by the MALDI manufacturer for bacterial identication (Bruker, Bremen, Germany)
Protocol #2: Protocol based on the method proposed by Yssouf etal. [9], with minor modications
Protocol #3: Protocol based on the method proposed by Müller etal. [22], with minor modications
Protocol #1 Protocol #2 Protocol #3
Selected mosquito body parts were placed in separate
microcentrifuge tubes, rinsed with 300 μL ultrapure
water and 900 μL ethanol, and centrifuged at
13,000 rpm for 2 min
Samples were decanted and treated with 10 μL of 70%
formic acid for 5 min at room temperature
Immediately after, samples were homogenized in the
tube with the help of a manual pestle with an addi‑
tional 10 μL of 100% acetonitrile and centrifuged at
13,000 rpm for 2 min
A small volume of supernatant was pre‑mixed
with equal volume of 10 mg/mL α‑cyano‑4‑
hydroxycinnamic acid (HCCA) matrix and 1 μL of the
mix was quickly placed in its respective target well in
triplicate
Selected mosquito body parts were rinsed with
distilled water and dried with paper
Samples were immediately homogenized with
the help of a manual pestle in 20 μL of 70%
formic acid and 20 μL of 100% acetonitrile and
incubated for 1 h
Samples were vortexed for 15 s, centrifuged at
13,000 rpm for 2 min and a small volume of the
supernatant was pre‑mixed with equal volume
of 10 mg/mL HCCA before adding 1 μL of the
mix it to the target well in triplicate
Selected mosquito body parts were
homogenized with the help of a
manual pestle in 20 μL of 10% formic
acid, pre‑mixing with 1.5 × volume of
sinapinic acid matrix, and centrifuged
at 13,000 rpm for 2 min
1 μL of supernatant was immediately
added to its respective target well in
triplicate
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Page 4 of 12
Loaizaetal. Malar J (2019) 18:95
of the methodology, the best extraction protocol and
the section of the mosquito body and sex with the high-
est protein signal and consistency were used. e goal
here was to determine if different Neotropical Anoph-
eles species, non-vectors and vectors of human Plas-
modium, had specific protein profiles generated with
MALDI that could be used for rapid and accurate iden-
tification purposes.
MALDI mass spectrometry parameters
e mass spectrometer used for the measurements was
an UltrafleXtreme III (Bruker Daltonics, Bremen, Ger-
many) equipped with a MALDI source, a time-of-flight
(TOF) mass analyzer, and a 2 KHhz Smartbeam-II
neodymium-doped yttrium aluminum garnet (Nd:YAG)
solid-state laser (λ = 355nm) used in positive polariza-
tion mode. All spectra were acquired with an automa-
tized script in the range of 2000 to 20,000m/z in linear
mode for the detection of the most abundant proteins.
Every spectrum represents the accumulation of 5000
shots with 300 shots taken at a time, and the acquisi-
tion was done in random-walk mode with a laser power
in the range of 50 to 100% (laser attenuation at 20%). To
promote the accuracy of the identification algorithms,
the spectra collected with the automatic script had to
include at least one peak with a minimum intensity of
3500 arbitrary units [a.u] as a stringent parameter of
quality to be considered “good quality” spectra. e
software FlexAnalysis (Bruker) was used to analyse
the spectra initially and to evaluate number of peaks,
peak intensity and perform simple spectra comparisons
to visually inspect for differences in dominant peaks
that would suggest possible classification into discrete
taxa. All samples were placed and measured on three
individual target wells with spectra from three techni-
cal replicates collected per well.
Data analysis, statistics andclustering algorithms
For routine mass spectra statistical analysis, including
two-dimensional (2D) peak distributions and principal
component analysis (PCA), the program ClintProTools
(Bruker) was used. Individual sample spectra were pre-
processed using smoothing and baseline subtraction
functions, and three-dimensional (3D) plots were gener-
ated to display unsupervised clustering at the subgenera
and species levels based on the most abundant protein
spectra. However, complete classification of spectra from
the field-collected mosquitoes could not be achieved
with the manufacturer’s software because reference
library entries that conformed to the quality standards of
the application could not be created.
For more stringent and comprehensive data clustering
and identification, a custom-made Linear Discriminant
Analysis (LDA) quantitative approach was implemented
using the software MATLAB® (MathWorks, Natick,
MA, USA). Given the size of the samples, a dimensional-
ity reduction stage was implemented using PCA as well.
Both approaches have been used in identification in the
context of face recognition [27, 28], and are established
methods used in spectral classification in the context of
mass spectrometry [29, 30].
Let the training set of the samples be Γ1, Γ2, Γ3,
…, ΓM1, ΓM. e average sample is defined as
=
1
M
M
i=1
Ŵ
i
. Each sample differs from the average
sample by the vector Φi = Γi Ψ. Given the mean-cen-
tered sample matrix
A=[1,2,3,...,M1,M]
,
the covariance matrix
C
=
1
M
M
n=1
n
T=AA
T
was
calculated. e eigenvectors of this covariance matrix
correspond to a set of orthonormal vectors that form a
basis to represent the data with a reduced dimension-
ality. A previously published approach [28] was used
to calculate indirectly the first M eigenvectors of the
matrix C, by estimating the eigenvectors of the matrix
L = ATA, reducing the memory and computational
requirements of this procedure.
PCA-based identification consists in using the projec-
tion of the sample in the eigenvectors to calculate a set of
coefficients
ωk
=u
T
k
[Ŵ],k=1, 2, 3, ...,M
<M to
describe each sample as a vector
=
ω
,ω
,...,ω
e average of the vectors describing the samples of the
training set of a given class was used to represent the
class in the new basis. en, to identify a test sample, the
Euclidean distance between the vector Ω describing the
test sample and the vectors describing each class were
calculated. e class with the minimum distance with
respect the test sample was assigned to the test sam-
ple. e PCA provides basis vectors that correspond to
the direction of maximal variance in the sample space.
In other words, using maximal variance as an unsuper-
vised parameter for clustering, the test samples are then
compared to the classes created with the information of
mosquito species that were identified morphologically; if
the distance between the test sample vector and the cor-
rect class (i.e., mosquito species) vector was the smallest
one, this was considered a positive identification. In the
other hand, LDA considers class information to provide
a basis that best discriminates the classes [27]. e LDA
can be applied over the data set expressed in terms of the
coefficients obtained by the PCA. us, PCA reduces the
dimensionality of the data, and the LDA provides super-
vised classification.
e LDA basis vectors
Wopt =[w
1
w
2
w
3
...wP]
are
obtained by calculating the matrix that maximizes the
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Loaizaetal. Malar J (2019) 18:95
ratio |W
T
SBW|
|W
T
SWW|
, where SB and SW are the between-class
scatter matrix and the within-class scatter matrix, respec-
tively. is new set of vectors maximizes the distance
between class means and minimizes the class variation.
For test sample identification, a similar Euclidean dis-
tance approach was implemented, as explained for the
PCA case. us, in this case using the between- and
within-class scatter ratio vectors as supervised parame-
ters for clustering, the test samples are compared to the
LDA basis vectors that contain the information of mos-
quito species that were identified morphologically; if the
distance between the test sample vector and the correct
class (i.e., mosquito species) vector was the smallest one,
this was considered a positive identification. e perfor-
mance of the LDA approach was tested using Monte
Carlo cross validation over 500 iterations. For each itera-
tion, the data is split randomly in 80% of the samples for
training and 20% of samples for testing, for each species.
For such implementation, the first 50 vectors or compo-
nents from the PCA stage were used, which after being
projected for the LDA algorithm, also generated a 50
components data set. is number of components was
chosen after a performance analysis using a Monte Carlo
approach. is number provided the best identification
rates. e total data set consists in 826 spectral samples
of 12 species.
Results
Sample preparation andoptimization
e three protein extraction protocols used herein were
relatively simple and, in general, involved a combina-
tion of formic acid and organic solvent to solubilize the
proteins present in each sample and facilitate their ioni-
zation (Table 1). Protocol #2 was the most time-con-
suming because of one washing step with water and 1-h
period to incubate the sample. Protocol #1 was slightly
faster than Protocol #2 since it did not involve the incu-
bation of the sample; however, it was the most labor
intensive due to one additional washing step with etha-
nol plus additional decantation and centrifuging steps.
Protocol #3 involved only two steps, was the fastest and
least labor intensive of the three protocols (Table1). e
two rinsing steps of Protocol #1 seemed to help reduce
noise in the spectra and improve repeatability of each
spectrum. A critical step in extraction protocol #1 was
the homogenization of the samples. Physical homog-
enization with a manual stainless-steel pestle tool and a
routing movement technique was used. Here the applied
force and time of homogenization were important to
obtain good quality spectra, probably due to the reduced
size and weight of the samples (e.g., one leg). Protocol #1
provided robust results during preliminary examinations
despite being the most labor intensive; thus, the influence
of different body sections and the sex of mosquitoes in
the mass spectra was analysed with protocol #1 only.
e different body sections of the mosquito presented
specific and repeatable mass spectra regardless of spe-
cies being analysed. In general, and across mosquito spe-
cies the head and the thorax were the most signal-rich
parts analysed. However, the legs, divided into anterior,
middle and posterior pairs, showed more robust and
repeatable signals (Additional file 3). Moreover, similar
results were found for A. albimanus, A. aegypti and A.
albopictus mosquitoes when using only one leg, so this
Table 2 Description ofsamples subjected toanalysis withtheMALDI mass spectrometry procedure
(a) = Río Indio, Colón; (b) = Jaqué, Darién; (c) = Quebrada Hilo, Bocas del Toro; (d) = La Miel, Puerto Obaldía, Darién; (e) = Finca 51, Guabito, Changuinola, Bocas del
Toro; (f) = Achiote, Colón; (g) = El Coco, Darién. Nys = Nyssorynchus; An = Anopheles; Ker = Kertezia. More information about these localities can be obtained from
gures and maps in references [4, 6, 7]
Mosquito species # ofspecimens Locality code # ofexpected
spectra # ofobtained
spectra MALDI good
spectra (%)
Anopheles (Nys) albimanus 51 a–g 153 119 78
Anopheles (An) apicimacula 40 b, d, g 120 110 92
Anopheles (Nys) aquasalis 19 c, d 57 56 98
Anopheles (Nys) darlingi 14 b, g 42 40 95
Anopheles (An) malefactor 13 b, d, g 39 39 100
Anopheles (Nys) nuneztovari 66 b, g 198 192 97
Anopheles (An) pseudopunctipennis 15 b, g 45 45 100
Anopheles (An) punctimacula 32 b, d, g 96 81 84
Anopheles (Nys) strodei 16 e 48 48 100
Anopheles (Nys) triannulatus 9 a, f 27 26 96
Anopheles (Ker) neivai 10 c, f 30 24 80
Chagasia bathana 15 f 45 45 100
Total 300 7 900 825 92
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Loaizaetal. Malar J (2019) 18:95
sample preparation was chosen to minimize the amount
of voucher used. Another question being evaluated was
if differences in the sex of the mosquito could affect the
spectra from the legs. e legs of male and female speci-
mens of A. albimanus, A. aegypti, A. albopictus were
compared and no evident differences were found in their
spectra due to sex (Additional file4). Given the repeat-
ability and lower risk of spectra variations with the legs,
and the presence of three pairs of legs per individual
that could serve as technical replicates for future experi-
ments, all further evaluations with field-collected sam-
ples of Anopheles were performed using female mosquito
legs only.
MALDI mass spectrometry toclassify eld‑collected
Anopheles species
e mass spectra of field-collected Anopheles mosqui-
toes, in general, had lower quality than that of labora-
tory-reared mosquitoes in terms of intensity of the signal
and signal-to-noise ratio, possibly due to contaminants
that were not removed during the rinsing steps of the
extraction protocol, which could have suppressed the
ionization of certain molecules or introduced noise in the
spectra (Table2). e percentage of good quality spec-
tra acquired from the prepared specimens in automatic
mode with the MALDI mass spectrometer ranged from
78% for A. albimanus to 100% for several of the species,
including Anopheles malefactor, Anopheles pseudopunc-
tipennis, Anopheles strodei and Chagasia bathana. All
biological specimens of Anopheles mosquitoes evaluated
in this study were capable of generating good quality
spectra (Table 2) and the specimens within each spe-
cies showed consistently similar protein profiles after
analysis with the MALDI technique, regardless of their
taxonomic subgenera, collection date and/or sampling
location. Mean protein spectra for Anopheles species dif-
fered visually among taxa and the differences appeared
to be related to their phylogenetic relationships (Fig.1).
For example, species within the subgenus Nyssorynchus
of Anopheles were more similar among them in terms
of pick number and position, than with other taxa from
a different subgenus. Nonetheless, somehow seemingly
closely related species, such as Anopheles punctimacula
and Anopheles malefactor within the Arribalzagia Series
of the subgenus Anopheles, depicted reasonably distinct
protein spectra that motivated the pursue of clustering
algorithms for their identification (Fig.1).
Distinct mass spectra profiles between morphologi-
cally-identified Anopheles species could be classified by
an unsupervised PCA algorithm to identify specimens.
e quantitative performance of the PCA algorithm
was assessed per species (Table 3), and visually con-
firmed with the clustering exhibited in 3D plots (Fig.2).
e PCA global positive identification rate was 89.83%,
with 7 out of 12 species having higher than 90% positive
identification rate. For visualization purposes in the PCA
scores plots, all species that were morphologically iden-
tified within the Anopheles or Nyssorynchus subgenera
were separately compared against Kertezia and Chagasia,
for which there was only one species in each. ree of
the species in the Anopheles subgenus belonged to the
Arribalzagia Series within this same subgenus as well
(i.e., Anopheles apicimacula, A. malefactor and A. punc-
timacula). e PCA 3D graph showed that each spe-
cies separated in well-defined clusters, and the distance
among clusters seemed to be related to the phylogenetic
relationships as evidenced by the clear separation from
the specimens of Chagasia bathana (Fig. 2a, b). All the
subgenera together were also compared using only two
species from each Anopheles and Nyssorynchus subgen-
era, for visualization purposes. Again, the spectra from
specimens of each species clearly clustered together, with
reasonable overlap between groups (Fig.2c, d).
In addition, LDA analysis for all 12 species was per-
formed using a Monte Carlo simulation with 500 itera-
tions to optimize training and cross-validation prediction
success rates (Fig.3; Table3). From all morphologically-
identified species, a training set with 80% of the samples
was randomly selected (Fig. 3a) and the other 20% was
used as a test set (Fig.3b), and this process was repeated
in each iteration. Global and class positive identification
rates were calculated to establish the classification capac-
ity of the algorithm (Table3). e positive identification
rate corresponds to the percent ratio between positive
identifications performed by the algorithm and the real
positive cases in the data. e global positive identifica-
tion rate obtained with the LDA was 93.33% (Table3,
Fig.3b), with a range that went from 100% (best score
possible) for Anopheles neivai and Chagasia bathana,
to 67.88% for A. malefactor. For visualization purposes,
the LDA representation of the Nysorrynchus (Fig.3c) or
Anopheles (Fig.3d) subgenera compared against the sub-
genera Kertezia and Chagasia (one species each) were
also plotted. e LDA clustering plots show that when
comparing species from different subgenera and even
within a particular subgenus, the separation between
specimens from different species is evident.
Discussion
Addressing thelimitations ofprevious studies
withtheMALDI
Proof of concept with the MALDI mass spectrometry
to examine species boundaries among arthropod vec-
tors of diseases has been well established before in ticks
(Ixodidae—Rhipicephalus) [16, 18], fleas (Pulicidae—
Ctenocephalides) [17], tsetse flies (Glossina spp.) [19],
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Loaizaetal. Malar J (2019) 18:95
Fig. 1 Baseline corrected and smoothed spectra for 11 species of mosquitoes in the genus Anopheles plus Chagasia bathana. Mayor peaks and
their molecular weights are annotated in the range of 2000 to 20,000 m/z for all species
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Loaizaetal. Malar J (2019) 18:95
sandflies (Psychodidae—Phlebotomus) [21, 31], biting
midges (Ceratopogonidae—Culicoides) [32] and mos-
quitoes (Culicidae) [10, 20, 2225]. However, many of
the experiments conducted up to now with the MALDI
involved laboratory-reared specimens and few species
or geographically discrete specimens of the same spe-
cies. Also, with some recent exceptions [9, 10, 2325],
full arthropod bodies were largely used in their protocols,
leaving no morphological vouchers for trial confirmation
and replication. Moreover, some of these publications
employed fairly distinct sample processing protocols,
thus making it difficult to decide about their appropriate-
ness and usefulness to study different arthropod group-
ings. Different methodologies to handle samples with
the MALDI mass spectrometry might result in different
outcomes, yet few published studies have evaluated the
influence of these differences on the resulting protein
spectra.
Here, a methodology was adjusted to use mosquitoes
of the same sex (i.e., only females) that were processed
for a specific body part (e.g., only legs) and with the
best protein extraction protocol based on comparisons
assumed on initial experiments using lab-colonized mos-
quitoes (i.e., Protocol #1). e MALDI mass spectrom-
etry technique could also be used effectively and timely
to discriminate among field-collected female individuals
of various Neotropical Anopheles species using only one
leg, while maintaining good signal robustness. e use of
legs to generate protein spectra from ticks and mosqui-
toes with the MALDI has been successfully accomplished
before [9, 10, 16, 2325], yet so far this approach has not
been used to classify samples of Neotropical Anopheles
species, nor has it been applied to field collected speci-
mens that were stored in silica gel.
Considering that one of the objectives of this study
was to find the smallest portion of the mosquito that
contained enough identifiable information in order to
preserve the specimen voucher for other molecular eco-
epidemiological assays, the results found with only one of
the legs per specimen are very attractive due to the pos-
sibility of keeping almost the entire insect body to inves-
tigate phylogenetic relationship, pathogen infection rate,
and identification of host blood type. Nevertheless, the
intensity of the spectra collected with MALDI may be
decreased when working with field-collected samples and
such limited amount of biological material for homog-
enization. Still, in this study 92% of the analysed matrix-
sample spots offered spectra with high-enough intensity
to be picked up by the automatized script (e.g., 825 out
of 897 spots from the three technical replicates per speci-
men), and only 3 of 12 tested species had a spectra col-
lection rate below 90%. Since the groups with the lower
spectra success rate included some of the more abundant
species such as A. albimanus (78%), A. punctimacula
(84%) and A. neivai (80%), and were equally likely across
different localities and sampling dates, the lower spectra
collection rate could potentially be due to degradation of
some samples under unfavorable storage condition, fail-
ure to load samples successfully in the metal plate of the
MALDI or contamination from the field. However, the
procedure allows researchers to try again several times
by using any of the remaining legs of the mosquito, thus
offering a practical and realistic way around this problem.
Future studies will have to test additional conservation
methods and determine if preserving samples in silica
gel was the cause of low success rates in obtaining the
expected number of spectra overall and per species.
Table 3 Performance ofPCA andLDA clustering algorithms
Species name PCA positive
identication rate (%) LDA positive
identication rate (%) Spectra
perclass # Training
elements # Test elements
Anopheles albimanus 91.48 97.40 119 95,000 24,000
Anopheles apicimacula 94.54 96.30 110 88,000 22,000
Anopheles aquasalis 99.99 99.90 56 44,000 12,000
Anopheles darlingi 95.65 99.30 40 32,000 8000
Anopheles malefactor 71.81 67.88 39 31,000 8000
Anopheles nuneztovari 81.47 86.73 192 153,000 39,000
Anopheles pseudopunctipennis 100.00 99.99 45 36,000 9000
Anopheles punctimacula 86.55 95.16 81 64,000 17,000
Anopheles strodei 88.75 93.63 48 39,000 10,000
Anopheles triannulatus 88.48 90.73 26 20,000 6000
Anopheles neivai 100.00 100.00 24 19,000 5000
Chagasia bathana 100.00 100.00 45 36,000 9000
Global 89.83 93.33 825 657,000 169,000
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Loaizaetal. Malar J (2019) 18:95
A way aroundworking withoutareference library
ofprotein spectra
e conventional MALDI biotyper approach for spe-
cies identification uses a reference library database of
laboratory-reared and well-characterized species-specific
protein spectra plus computational software from the
vendor to compare unknown spectra to those in the ref-
erence library. e program generates a degree of simi-
larity between sample spectra and the reference library,
and gives a simplified score ranging from 0.0 to 3.0, in
which any score above or equal to 2.7, represents a per-
fect match between a sample spectrum and a particu-
lar library spectrum and 2.3 can be used as a minimum
threshold for an accurate identification at the species
level. is methodology has been very successful for
clinical studies involving pathogenic bacteria to humans
because they are easy to cultivate in the laboratory and
their colony-forming units offer robust and repeatable
signals [33]. However, to build a reference library with
fresh and well-curated Anopheles species requires high-
quality, extremely consistent spectra from mosquitoes
collected in the field as immature stages and lab-reared in
the insectary, which is complicated to accomplish either
due to difficulties in field collecting larvae of some spe-
cies or laboratory-rearing them in the insectary [34]. To
date, only partial reference libraries with protein spectra
from a mixture of laboratory-reared and field-collected
mosquitoes have been built with mixed quality standards,
forcing the use of alternating lower threshold scores for
species identification of 1.8 [9, 10, 2325] or as low as 1.3
in recent studies [35]. In addition, none of these studies
have included Neotropical Anopheles species.
e quality of the spectra from the field-collected mos-
quitoes analysed in this study was lower than expected,
Fig. 2 Principal component analysis (PCA) of individual observations plotted against first, second and third principal components (PC). a All species
belonging to the Anopheles subgenus of Anopheles, including three of them belonging to the Series Arribalzagia within this same subgenus as well
(i.e., Anopheles apicimacula, A. malefactor and Anopheles punctimacula), were clustered in comparison to the Kertezia and Chagasia subgenera. b A
similar analysis was performed for the Nyssorynchus subgenus compared to the same Kertezia & Chagasia species and c, d with all four subgenera
together in the same analysis, picking only two species of each of the subgenera that had more abundant species. Different colors represent
different species
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Loaizaetal. Malar J (2019) 18:95
requiring the use of other statistical techniques for iden-
tification. Mass fingerprinting for the identification of
field-collected specimens that do not exist in a refer-
ence library or for those whose reference spectra cannot
be generated, requires alternative approaches that can
be developed to detect distinctive features in the spec-
tra of unknown samples. To address this shortcoming,
smoothed and baseline corrected spectra were produced
from field-collected samples of 11 species of mosqui-
toes in the genus Anopheles plus Chagasia bathana
and compared against the mean spectra from the same
field samples as a self-curated reference library. Further,
a combination of unsupervised (PCA) and supervised
mathematical algorithms (LDA) were used to clas-
sify mass spectra of field-collected Anopheles with high
consistency.
In general, PCA outcomes were less discriminant and
robust than LDA, still PCA discriminated among Anoph-
eles species from different subgenera with almost 90%
accuracy and consistency. LDA was able to classify all
12 species of mosquitoes together with validation and
cross-validation scores above 93%, both between and
within subgenera. is included samples from seven
localities across the entire country of Panama, includ-
ing vectors and non-vectors of Plasmodium. Evidently,
the clustering algorithm was more accurate for mosquito
species that were phylogenetically distinct from the rest
(i.e., Kertezia and Chagasia subgenera), with 100% suc-
cess rate in these cases; while the success rate decreased
for more closely related species (i.e., A. malefactor, from
the Arribalzagia Series). Still, the global success rate
was 93.33%, which is reasonably precise. erefore, due
to its supervised nature LDA was able to identify field-
collected Anopheles species without the need of a refer-
ence library of species-specific protein spectra, and with
higher resolution and discriminant power than PCA.
Conclusion
A methodology was developed that allows the identifi-
cation of field-collected mosquitos from the Anopheles
genus without prior establishment of a reference library
Fig. 3 Linear discriminant analysis (LDA) applied to mosquito species of the subgenera Anopheles, Nyssorhynchus, Anopheles (Kertezia) neivai and
Chagasia bathana. a Plot of the training set for all species projected over the first three components of the LDA. b Plot of the test set for all species
projected over the first three components of the LDA. c Plot of the test set in the Nyssorynchus subgenera compared to the Anopheles (Kertezia)
neivai and Chagasia bathana species, projected over the first three components of the LDA. d Plot of the test set in the Anopheles subgenera
compared to the Anopheles (Kertezia) neivai and Chagasia bathana species projected over the first three components of the LDA. These 3D plots
represent only one of the 500 Monte Carlo iterations performed with the algorithm. The algorithm had a 93.33% global positive identification rate
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Loaizaetal. Malar J (2019) 18:95
of well-curated lab-reared mosquitoes. Prior scientific
work in Panama and elsewhere suggests that DNA bar-
codes occasionally fail to elucidate the evolutionary
relationships among closely related Anopheles species.
Although the number of mosquitoes analysed in this
study is still relatively low, the results show that the clas-
sification algorithms used here were capable of clustering
and identifying spectra from up to 12 different field-col-
lected mosquito species. In future studies, this MALDI
procedure will be tested to discriminate between geo-
graphically isolated populations/lineages of cryptic spe-
cies complexes such as A. punctimacula sensu lato (s.l.)
and Anopheles apicimacula s.l. is approach can be eas-
ily adapted and applied more broadly to other tropical
regions of the world where Anopheles species diversity is
high and morphological species complexes do exist.
Additional les
Additional le1. Optical micrographs of three mosquito species: (A, B
and C) Images of lab‑reared Anopheles albimanus (A), Aedes aegypti (B) and
Aedes albopictus (C) (Left to right in that order) used for the optimization of
the sample preparation protocol and MALDI mass spectrometry analysis.
Additional le2. Magnified images of different body parts of Aedes
albopictus used to generate protein spectra with the MALDI mass spec‑
trometry approach.
Additional le3. Protein spectra generated from different body parts
of female mosquitoes (On the left side). Optical micrographs of the
head, thorax, abdomen, wings and anterior, middle and posterior legs of
laboratory‑reared Aedes albopictus (On the right side, in that order).
Additional le4. Comparison of protein spectra generated from the
middle legs of males (Top) and females (Bottom) of Anopheles albimanus,
Aedes aegypti and Aedes albopictus mosquitoes.
Additional le5. Mosquito trapping methods: (A) Intersection trap; (B)
Shannon trap; (C) CDC Miniature Light trap; (D) Larvae collection and (E)
sample processing procedure used in the study.
Abbreviations
MALDI: matrix‑assisted laser desorption/ionization; PCA: principal component
analysis; LDA: linear discriminant analysis; DNA: deoxyribonucleic acid; INDI‑
CASAT: Institute for Scientific Research and High Technology Services; STRI:
Smithsonian Tropical Research Institute; SNI: National System of Investigation;
MHIRT: Minority Health and Health Disparities International Research Training
Program; NIH: National Institute of Health; UTP: Technological University of
Panama; TOF: time‑of‑flight; MINSA: Ministry of Health; MiAmbiente: Ministry
of Environment; SENAFRONT: National Border Protection Service; CDC: Center
for Disease Control.
Authors’ contributions
JRL and RAG designed and developed the experiments. JRL collected and
identified the mosquitoes. AA, NDC and JCR performed the tests with the
MALDI. JRL, JSG, FM, AG and RG analysed the data and produced the graphs.
JRL and RAG wrote the first draft of the paper and LM, LFL, MJM, JSG, and FM
contributed comments to subsequent versions on it. All authors read and
approved the final manuscript.
Author details
1 Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de Investi‑
gaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), City of
Knowledge, Panama 0843‑01103, Republic of Panama. 2 Smithsonian Tropical
Research Institute, Panama, Republic of Panama. 3 Programa Centroamericano
de Maestría en Entomología, Universidad de Panamá, Panama, Republic
of Panama. 4 College of Health Sciences, The University of Texas at El Paso, El
Paso, TX, USA. 5 Grupo de Investigación en Biotecnología, Bioinformática y
Biología de Sistemas, Centro de Producción e Investigaciones Agroindustriales,
Universidad Tecnológica de Panamá, Panama, Republic of Panama. 6 Grupo de
Investigación en Sistemas de Comunicaciones Digitales Avanzados, Facultad
de Ingeniería Eléctrica, Universidad Tecnológica de Panamá, Panama, Republic
of Panama. 7 ENSEIRB‑MATMECA‑Bordeaux INP, Talence, France. 8 Sam Noble
Oklahoma Museum of Natural History and Department of Biology, University
of Oklahoma, Norman, OK, USA. 9 Department of Biology, University of Mas‑
sachusetts Boston, Boston, MA, USA. 10 Centro de Neurociencias, INDICASAT
AIP, Panama, Republic of Panama.
Acknowledgements
Special thanks to Jose R. Rovira, Urbano Arrocha, Dagoberto Atencio, Rufino
Valdez and Cipriano Ayarza from the Panamanian Ministry of Health (MINSA),
vector control department, for assisting with mosquito collections during the
study. Also to Gilberto Eskildsen, Larissa Dutari, Gaudenia Mendoza, Jahzeel
Samaniego and Eric Alvarez from the University of Panama for helping with
logistic during several field trips. Special thanks to Aishwarya Sunderrajan,
from the Madras Institute of Technology, India, and Jonathan Kern, from
ENSEIRB‑MATMECA—Bordeaux INP, France, for their support generating the
final high‑resolution figures for the publication. Special thanks to the Panama‑
nian Ministry of Environment (MiAmbiente) for supporting scientific collecting
of mosquitoes across Panama. Finally, special thanks to the members of
SENAFRONT for offering protection while working in Darien, near the border
with Colombia.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Funding
Financial support for this work was provided by SENACYT through the
research grant GRID15‑002 to JRL, LM, JSG, LFD and RAG and NIH grant
5T37MD001376‑12 to College of Health Sciences, University of Texas at El Paso.
INDICASAT‑AIP, UTP and STRI provided additional economic and logistic sup‑
port. The SNI supports research activities by JRL (SNI 05‑2016 & SNI 157‑2017),
JSG, LM, LFD, FM and RAG (SNI 91‑2015 & SNI 146‑2017). RAG is also supported
by SENACYT grants FID14‑066, ITE15‑016.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
Received: 24 September 2018 Accepted: 12 March 2019
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... Several teams have built in-house databases to identify species of adults Anopheles by their MALDI-TOF spectra. Some of them used the legs to minimize the amount of material from specimen vouchers [10][11][12][13][14], whereas some other studies used the cephalothorax [15,16]. Consequently, there is no general consensus regarding the optimal anatomic part to be used. ...
... The spectra from the legs exhibited the smaller number of peaks of high intensity, showing that the protein content was less diverse than for the head and thorax. Previous studies concluded that legs provided sufficient protein material to give reproducible and specific mass spectra [10][11][12][13][14]. However, a recent study reported that the using of less than four legs could compromise the MALDI-TOF MS identification of mosquito species, showing that at least four legs are required to get sufficient protein material [25]. ...
... In addition, one of the previous studies observed Fig. 6 Distribution of identification log(scores) according to number of spots per specimen using panel A versus database 2. The best log (score) was recorded according to the using of one, two, three or four spots of protein extract of legs (a) and head (b). The results of combinations of spots were analysed chronologically from the first to the fourth sample of protein extract deposited onto the target plate that the quality of legs spectra from field-caught Anopheles was lower than that from colony specimens, with a decreased intensity [14]. This suggests a possible protein degradation of the legs from field-caught specimens. ...
Article
Full-text available
Background Anopheles species identification is essential for an effective malaria vector control programme. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been developed to identify adult Anopheles species, using the legs or the cephalothorax. The protein repertoire from arthropods can vary according to compartment, but there is no general consensus regarding the anatomic part to be used. Methods To determine the body part of the Anopheles mosquitoes best suited for the identification of field specimens, a mass spectral library was generated with head, thorax with wings and legs of Anopheles gambiae , Anopheles arabiensis and Anopheles funestus obtained from reference centres. The MSL was evaluated using two independent panels of 52 and 40 An. gambiae field-collected in Mali and Guinea, respectively. Geographic variability was also tested using the panel from Mali and several databases containing added specimens from Mali and Senegal. Results Using the head and a database without specimens from the same field collection, the proportion of interpretable and correct identifications was significantly higher than using the other body parts at a threshold value of 1.7 (p < 0.0001). The thorax of engorged specimens was negatively impacted by the blood meal after frozen storage. The addition of specimens from Mali into the database significantly improved the results of Mali panel (p < 0.0001), which became comparable between head and legs. With higher identification scores, the using of the head will allow to decrease the number of technical replicates of protein extract per specimen, which represents a significant improvement for routine use of MALDI-TOF MS. Conclusions The using of the head of Anopheles may improve the performance of MALDI-TOF MS. Region-specific mass spectrum databases will have to be produced. Further research is needed to improve the standardization in order to share online spectral databases.
... d'anophèles. La majorité d'entre elles utilisaient le matériel protéique issu des pattes, comme pour l'ensemble des Culicidae(Diarra et al., 2019;Lawrence et al., 2018;Loaiza et al., 2019;Raharimalala et al., 2017;Rakotonirina et al., 2020;Tandina, Niare, et al., 2018;Yssouf et al., 2014Yssouf et al., , 2013, mais d'autres proposaient d'exploiter spécifiquement le céphalothorax dans le cas des anophèles(Mewara et al., 2018;Müller et al., 2013). Jusqu'ici, aucun consensus n'a été établi au regard de la partie anatomique à privilégier. ...
... Jusqu'ici, aucun consensus n'a été établi au regard de la partie anatomique à privilégier. Toutefois, les dernières études ont montré une sensibilité importante des pattes aux conditions de capture, de transport et de stockage(Diarra et al., 2019;Loaiza et al., 2019;Rakotonirina et al., 2020). Par ailleurs, l'utilisation du céphalothorax d'anophèles capturés sur le terrain et potentiellement gorgés a fait suspecter de possibles contaminations lors des dissections à partir du sang contenu dans l'abdomen(Müller et al., 2013). ...
Thesis
Full-text available
Les programmes de contrôle vectoriel sont une priorité stratégique dans le contrôle du paludisme et des autres maladies à transmission vectorielle. Toutefois, les outils entomologiques de caractérisation des arthropodes vecteurs sont limités et difficiles à mettre en œuvre sur le terrain. Ainsi, l’objectif de cette thèse était la mise au point de nouveaux outils protéomiques de surveillance des arthropodes vecteurs grâce à la spectrométrie de masse MALDI-TOF (désorption et ionisation assistée par une matrice avec détection en temps de vol). Les travaux ont validé l’outil MALDI-TOF pour identifier les espèces de phlébotomes de Guyane et d’anophèles de Guinée et du Mali. Pour les spécimens d’Anopheles stephensi d’élevage, les réseaux de neurones artificiels couplés au MALDI-TOF reconnaissaient des motifs spectraux liés à la biologie des anophèles : l’âge, les antécédents de repas sanguin et l’infection par Plasmodium berghei. Les études futures devront valider les nouvelles approches à plus grande échelle à partir de spécimens collectés sur le terrain. Une application en ligne, développée à Sorbonne Université pour l’identification MALDI-TOF en microbiologie, facilitera l’utilisation pour la surveillance vectorielle en partageant des banques de spectres d’arthropodes. Enfin, les approches bio-informatiques pourront améliorer les performances et fournir de nouvelles applications.
... MALDI-TOF MS biotyping emerged recently as a relevant alternative solution, to dispense with morphological and molecular biology identification tools [18]. This MS biotyping tool was successfully applied for the identification of mosquitoes at the adult [20,29] but also at the immature stages [21,22]. Nevertheless, the rates of correct identification at the pupal stages were lower than at late-instar stages using MALDI-TOF MS biotyping [22,30]. ...
... In another study [32], the comparison of protein patterns of pupal cuticles and larval head capsules from An. gambiae on SDS-PAGE gel revealed distinct profiles with a major band of low molecular weight for larval samples. Likewise, the specificity of MS spectra according to the body part used from the same species was recently reported [20,29]. The advantages of being able to query independently protein profiles from exuviae of fourth-instar larvae and pupae from the same specimens against the reference MS spectra DB, allow a double control for the identification of the mosquito. ...
Article
Full-text available
Background: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) biotyping is an innovative strategy, applied successfully for the identification of numerous arthropod families including mosquitoes. The effective mosquito identification using this emerging tool was demonstrated possible at different steps of their life-cycle, including eggs, immature and adult stages. Unfortunately, for species identification by MS, the euthanasia of the mosquito specimen is required. Methods: To avoid mosquito euthanasia, the present study assessed whether aedine mosquitoes could be identified by MALDI-TOF MS biotyping, using their respective exuviae. In this way, exuviae from the fourth-instar and pupal stages of Aedes albopictus and Aedes aegypti were submitted to MALDI-TOF MS analysis. Results: Reproducible and specific MS spectra according to aedine species and stage of exuviae were observed which were objectified by cluster analyses, composite correlation index (CCI) tool and principal components analysis (PCA). The query of our reference MS spectra database (DB) upgraded with MS spectra of exuviae from fourth-instar larvae and pupae of both Aedes species revealed that 100% of the samples were correctly classified at the species and stage levels. Among them, 93.8% (135/144) of the MS profiles reached the threshold log score value (LSV > 1.8) for reliable identification. Conclusions: The extension of reference MS spectra DB to exuviae from fourth-instar and pupal stages made now possible the identification of mosquitoes throughout their life-cycle at aquatic and aerial stages. The exuviae presenting the advantage to avoid specimen euthanasia, allowing to perform complementary analysis on alive mosquitoes.
... However, in metazoan identification it is still in its infancy. Nevertheless, successful species identification of specimens has been achieved from a large variety of taxonomic groups such as different crustaceans (Bode et al., 2017;Hynek et al., 2018;Kaiser et al., 2018;Kürzel et al., 2022;Laakmann et al., 2013;Paulus et al., 2022;Rossel & Martínez Arbizu, 2018b, fish (Maász et al., 2017;Mazzeo et al., 2008;Rossel et al., 2020;Volta et al., 2012), cnidarians (Holst et al., 2019;Korfhage et al., 2022), molluscs (Hamlili, Thiam, et al., 2021;Wilke et al., 2020) and a large variety of insects, preferentially those which are potential disease vectors (Dieme et al., 2014;Hasnaoui et al., 2022;Loaiza et al., 2019;Mathis et al., 2015;Nabet et al., 2021;Nebbak et al., 2017;Yssouf et al., 2014). ...
Article
Full-text available
Species identification is pivotal in biodiversity assessments and proteomic fingerprinting by MALDI‐TOF mass spectrometry has already been shown to reliably identify calanoid copepods to species level. However, MALDI‐TOF data may contain more information beyond mere species identification. In this study, we investigated different ontogenetic stages (copepodids C1‐C6 females) of three co‐occurring Calanus species from the Arctic Fram Strait, which cannot be identified to species level based on morphological characters alone. Differentiation of the three species based on mass spectrometry data was without any error. In addition, a clear stage‐specific signal was detected in all species, supported by clustering approaches as well as machine learning using Random Forest. More complex mass spectra in later ontogenetic stages as well as relative intensities of certain mass peaks were found as the main drivers of stage distinction in these species. Through a dilution series, we were able to show that this did not result from the higher amount of biomass that was used in tissue processing of the larger stages. Finally, the data were tested in a simulation for application in a real biodiversity assessment by using Random Forest for stage classification of specimens absent from the training data. This resulted in a successful stage‐identification rate of almost 90%, making proteomic fingerprinting a promising tool to investigate polewards shifts of Atlantic Calanus species and, in general, to assess stage compositions in biodiversity assessments of Calanoida, which can be notoriously difficult using conventional identification methods.
... Aside from microbiology, nowadays MALDI-TOF MS is also applied to fight food fraud [27], e.g. to detect mislabeling of sea food species [28][29][30][31][32][33][34][35], identify meat origin [36] or inspect milk adulteration [37]. Moreover, MALDI-TOF MS was tested in numerous studies to identify important disease vectors such as mosquitos, ticks and phlebotomine sand flies [38][39][40][41]. But it was also successfully applied in ecological studies [42][43][44] with high species identification accuracy based on reference libraries. ...
Article
Quantifying spawning biomass of commercially relevant fish species is important to generate fishing quotas. This will mostly rely on the annual or daily production of fish eggs. However, these have to be identified precisely to species level to obtain a reliable estimate of offspring production of the different species. Because morphological identification can be very difficult, recent developments are heading towards application of molecular tools. Methods such as COI barcoding have long handling times and cause high costs for single specimen identifications. In order to test MALDI-TOF MS, a rapid and cost-effective alternative for species identification, we identified fish eggs using COI barcoding and used the same specimens to set up a MALDI-TOF MS reference library. This library, constructed from two different MALDI-TOF MS instruments, was then used to identify unknown eggs from a different sampling occasion. By using a line of evidence from hierarchical clustering and different supervised identification approaches we obtained concordant species identifications for 97.5% of the unknown fish eggs, proving MALDI-TOF MS a good tool for rapid species level identification of fish eggs. At the same time we point out the necessity of adjusting identification scores of supervised methods for identification to optimize identification success. Significance Fish products are commercially highly important and many societies rely on them as a major food resource. Over many decades stocks of various relevant fish species have been reduced due to unregulated overfishing. Nowadays, to avoid overfishing and threatening of important fish species, fish stocks are regularly monitored. One component of this monitoring is the monitoring of spawning stock sizes. Whereas this is highly dependent on correct species identification of fish eggs, morphological identification is difficult because of lack of morphological features.
Article
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Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used for proteomic fingerprinting and taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high-quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions , or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrome-try as a rapid and effective method to complement other well-established techniques for
Article
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Background Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology (MALDI-TOF MS) is an innovative tool that has been shown to be effective for the identification of numerous arthropod groups including mosquitoes. A critical step in the implementation of MALDI-TOF MS identification is the creation of spectra databases (DB) for the species of interest. Mosquito legs were the body part most frequently used to create identification DB. However, legs are one of the most fragile mosquito compartments, which can put identification at risk. Here, we assessed whether mosquito thoraxes could also be used as a relevant body part for mosquito species identification using a MALDI-TOF MS biotyping strategy; we propose a double DB query strategy to reinforce identification success. Methods Thoraxes and legs from 91 mosquito specimens belonging to seven mosquito species collected in six localities from Guadeloupe, and two laboratory strains, Aedes aegypti BORA and Aedes albopictus Marseille, were dissected and analyzed by MALDI-TOF MS. Molecular identification using cox1 gene sequencing was also conducted on representative specimens to confirm their identification. Results MS profiles obtained with both thoraxes and legs were highly compartment-specific, species-specific and species-reproducible, allowing high identification scores (log-score values, LSVs) when queried against the in-house MS reference spectra DB (thorax LSVs range: 2.260–2.783, leg LSVs range: 2.132–2.753). Conclusions Both thoraxes and legs could be used for a double DB query in order to reinforce the success and accuracy of MALDI-TOF MS identification. Electronic supplementary material The online version of this article (10.1186/s13071-018-3157-1) contains supplementary material, which is available to authorized users.
Article
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Background: Accurate and rapid identification of dipteran vectors is integral for entomological surveys and is a vital component of control programs for mosquito-borne diseases. Conventionally, morphological features are used for mosquito identification, which suffer from biological and geographical variations and lack of standardization. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for protein profiling of mosquito species from North India with the aim of creating a MALDI-TOF MS database and evaluating it. Methods: Mosquito larvae were collected from different rural and urban areas and reared to adult stages. The adult mosquitoes of four medically important genera, Anopheles, Aedes, Culex and Armigerus, were morphologically identified to the species level and confirmed by ITS2-specific PCR sequencing. The cephalothoraces of the adult specimens were subjected to MALDI-TOF analysis and the signature peak spectra were selected for creation of database, which was then evaluated to identify 60 blinded mosquito specimens. Results: Reproducible MALDI-TOF MS spectra spanning over 2-14 kDa m/z range were produced for nine mosquito species: Anopheles (An. stephensi, An. culicifacies and An. annularis); Aedes (Ae. aegypti and Ae. albopictus); Culex (Cx. quinquefasciatus, Cx. vishnui and Cx. tritaenorhynchus); and Armigerus (Ar. subalbatus). Genus- and species-specific peaks were identified to create the database and a score of > 1.8 was used to denote reliable identification. The average numbers of peaks obtained were 55-60 for Anopheles, 80-100 for Aedes, 30-60 for Culex and 45-50 peaks for Armigeres species. Of the 60 coded samples, 58 (96.67%) were correctly identified by MALDI-TOF MS with a score > 1.8, while there were two unreliable identifications (both Cx. quinquefasciatus with scores < 1.8). Conclusions: MALDI-TOF MS appears to be a pragmatic technique for accurate and rapid identification of mosquito species. The database needs to be expanded to include species from different geographical regions and also different life-cycle stages to fully harness the technique for entomological surveillance programs.
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We develop a face recognition algorithm which is insensitive to large variation in lighting direction and facial expression. Taking a pattern classification approach, we consider each pixel in an image as a coordinate in a high-dimensional space. We take advantage of the observation that the images of a particular face, under varying illumination but fixed pose, lie in a 3D linear subspace of the high dimensional image space-if the face is a Lambertian surface without shadowing. However, since faces are not truly Lambertian surfaces and do indeed produce self-shadowing, images will deviate from this linear subspace. Rather than explicitly modeling this deviation, we linearly project the image into a subspace in a manner which discounts those regions of the face with large deviation. Our projection method is based on Fisher's linear discriminant and produces well separated classes in a low-dimensional subspace, even under severe variation in lighting and facial expressions. The eigenface technique, another method based on linearly projecting the image space to a low dimensional subspace, has similar computational requirements. Yet, extensive experimental results demonstrate that the proposed “Fisherface” method has error rates that are lower than those of the eigenface technique for tests on the Harvard and Yale face databases
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Background Malaria is still a major public health issue worldwide, and one of the best approaches to fight the disease remains vector control. The current methods for mosquito identification include morphological methods that are generally time-consuming and require expertise, and molecular methods that require laboratory facilities with relatively expensive running costs. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) technology, routinely used for bacterial identification, has recently emerged in the field of entomology. The aim of the present study was to assess whether MALDI-TOF MS could successfully distinguish Anopheles stephensi mosquitoes according to their Plasmodium infection status. Methods C57BL/6 mice experimentally infected with Plasmodium berghei were exposed to An. stephensi bites. For the determination of An. stephensi infection status, mosquito cephalothoraxes were dissected and submitted to mass spectrometry analyses and DNA amplification for molecular analysis. Spectra were grouped according to mosquitoes’ infection status and spectra quality was validated based on intensity and reproducibility within each group. The in-lab MALDI-TOF MS arthropod reference spectra database, upgraded with representative spectra from both groups (infected/non-infected), was subsequently queried blindly with cephalothorax spectra from specimens of both groups. Results The MALDI TOF MS profiles generated from protein extracts prepared from the cephalothorax of An. stephensi allowed distinction between infected and uninfected mosquitoes. Correct classification was obtained in blind test analysis for (79/80) 98.75% of all mosquitoes tested. Only one of 80 specimens, an infected mosquito, was misclassified in the blind test analysis. Conclusions Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry appears to be a promising, rapid and reliable tool for the epidemiological surveillance of Anopheles vectors, including their identification and their infection status.
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Background: Taxonomy that utilizes morphological characteristics has been the gold standard method to identify mosquito species. However, morphological identification is challenging when the expertise is limited and external characters are damaged because of improper specimen handling. Therefore, we explored the applicability of mitochondrial cytochrome C oxidase subunit 1 (COI) gene-based DNA barcoding as an alternative tool to identify mosquito species. In the present study, we compared the morphological identification of mosquito specimens with their differentiation based on COI barcode, in order to establish a more reliable identification system for mosquito species found in Singapore. Methods: We analysed 128 adult mosquito specimens, belonging to 45 species of 13 genera. Phylogenetic trees were constructed for Aedes, Anopheles, Culex and other genera of mosquitoes and the distinctive clustering of different species was compared with their taxonomic identity. Results: The COI-based DNA barcoding achieved a 100% success rate in identifying the mosquito species. We also report COI barcode sequences of 16 mosquito species which were not available previously in sequence databases. Conclusions: Our study utilised for the first time DNA barcoding to identify mosquito species in Singapore. COI-based DNA barcoding is a useful tool to complement taxonomy-based identification of mosquito species.
Article
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Background: Mosquitoes transmit a wide range of human parasitic and viral diseases. In recent years, new techniques such as MALDI-TOF MS have been developed to identify mosquitoes at the species level, which is key for entomological surveys. Additionally, there is increasing interest in the mosquito microbiota and its role in vector capacity. Methods: The culturomics approach previously used in our laboratory to study human gut microbiota was applied to evaluate the midgut bacterial diversity of Anopheles gambiae (wild and laboratory strains), Aedes albopictus (wild and laboratory strains) and Culex quinquefasciatus (wild strains) in order to determine the influence of the environmental status on the midgut microbiota of the mosquitoes. Results: Mosquitoes collected in the field were accurately identified by MALDI-TOF MS analysis of their legs. Adult mosquito midgut microbiota was composed of four phyla, including Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The majority of the bacteria detected in the microbiota of mosquitoes were gram-negative and belong to the phylum Proteobacteria. MALDI-TOF MS identified for the first time a new bacterial species from An. gambiae midgut microbiota. Conclusion: In this study, the culturomics approach was found to be a reliable technique for exploring the diversity of the mosquito microbiota. MALDI-TOF MS was confirmed as a promising technique to identify mosquitoes collected in the field. Culturomics allowed the isolation of a new bacterial species not previously associated with mosquito vectors. The environment plays a role in the bacterial diversity of the microbiota, which could enable the development of new control strategies for mosquito-borne disease.
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From 2002–2005, Panama experienced a malaria epidemic that has been associated with El Niño Southern Oscillation weather patterns, decreased funding for malaria control, and landscape modification. Case numbers quickly decreased afterward, and Panama is now in the pre-elimination stage of malaria eradication. To achieve this new goal, the characterization of epidemiological risk factors, foci of transmission, and important anopheline vectors is needed. Of the 24,681 reported cases in these analyses (2000–2014), ~62% occurred in epidemic years and ~44% in indigenous comarcas (5.9% of Panama’s population). Sub-analyses comparing overall numbers of cases in epidemic and non-epidemic years identified females, comarcas and some 5-year age categories as those disproportionately affected by malaria during epidemic years. Annual parasites indices (APIs; number of cases per 1,000 persons) for Plasmodium vivax were higher in comarcas compared to provinces for all study years, though P. falciparum APIs were only higher in comarcas during epidemic years. Interestingly, two comarcas report increasing numbers of cases annually, despite national annual decreases. Inclusion of these comarcas within identified foci of malaria transmission confirmed their roles in continued transmission. Comparison of species distribution models for two important anophelines with Plasmodium case distribution suggest An. albimanus is the primary malaria vector in Panama, confirmed by identification of nine P. vivax-infected specimen pools. Future malaria eradication strategies in Panama should focus on indigenous comarcas and include both active surveillance for cases and comprehensive anopheline vector surveys.
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Background: Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine. Methodology/principal findings: Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M'sila where P. (Phlebotomus) papatasi was the only sand fly species detected. Conclusion: The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF MS analyses opens up new ways in the management of phlebotomine sand fly-borne diseases.
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Background: This study aims to describe the epidemiological and entomological factors associated with a recent malaria outbreak that occurred in 2012 in a socially marginalized population from Guna Yala Comarca in Panama. Methods: A descriptive and observational study was conducted by analysing demographic and epidemiological data from all malaria cases registered during 2012 in the Comarca Guna Yala, Panama. Malaria intensity indicators were calculated during the study period. Entomological evaluations were performed monthly, from October to December 2012, in the three communities that presented the most intense malaria transmission during the first semester of 2012. Anopheles breeding habitats were also characterized. Results: During the studied period, 6754 blood smears were examined (17.8 % of the total population), and 143 were confirmed as positive for Plasmodium vivax. A significant increase of malaria transmission risk indicators (API: 3.8/1000, SPR: 2.1 %) was observed in Guna Yula, when compared with previous years, and also in comparison with estimates from the whole country. Anopheles albimanus was the most abundant and widespread (877; 72.0 %) vector species found in the three localities, followed by Anopheles punctimacula (231; 19.0 %) and Anopheles aquasalis (110; 9.0 %). Three An. albimanus pools were positive for P. vivax, showing an overall pooled prevalence estimate of 0.014. Conclusions: Data analysis confirmed that during 2012 a malaria epidemic occurred in Guna Yala. Panama. This study provides baseline data on the local epidemiology of malaria in this vulnerable region of Panamá. This information will be useful for targeting control strategies by the National Malaria Control Programme.